| One of the prerequisites to the study of structure-function relationships of the kallikrein-like proteinases is the availability of a heterologous expression system wherein enzyme engineering can be performed. For this purpose, a full length rat tissue kallikrein cDNA was constructed from a partial kallikrein cDNA clone by oligonucleotide engineering. Recombinant rat tissue kallikrein was expressed both in E. coli and yeast as detected by direct radioimmunoassay and Western blot analysis. Kallikrein was purified to apparent homogeneity from E. coli and its identity confirmed by N-terminal amino acid sequencing. Both E. coli and yeast recombinant kallikreins displayed comparable properties with the native tissue kallikrein with respect to size, charge, susceptibility to inhibitors, immunological properties as well as specific kininogenase activity. Rat tonin cDNA was also expressed in E. coli as demonstrated by direct radioimmunoassay and Western blot analysis. We were unable to purify tonin in active form due to the inability to solubilize it.; A set of four kallikrein mutants, Pro219 deletion (P219del), the 34-38 loop mutation (YYFG(34-38)IN), Trp215 to Gly exchange (W215G), and the double mutant with Tyr99 to His and Trp215 to Gly exchange (Y99H:W215G) were created by site-directed mutagenesis in order to probe their functions in substrate binding. The mutant proteins were expressed in the above E. coli system at high levels and purified to homogeneity. Our kinetic study supports the hypothesis that the Tyr99-Trp215 hydrophobic interaction dictates the P2 affinity for a hydrophobic side-chain and suggests an important role for the 34-38 loop in P3 specificity. Our results have also established the critical role Pro219 plays in both substrate binding and efficient catalysis of the kallikrein-like enzymes.; As a first step toward understanding the physiological significance of tissue kallikreins, transgenic mice have been generated which carry the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter. Human tissue kallikrein was expressed in the pancreas, salivary gland, kidney, liver and spleen, and it reached high levels in the circulation. Both lines of transgenic mice developed hypotensive phenotype with their systolic blood pressure significantly lowered compared with control mice. Administration of a potent kallikrein inhibitor restored the blood pressure of the transgenic mice. The results raise the possibility of tissue kallikrein as a powerful modulator of blood pressure and they also provide an excellent animal model for controlling blood pressure through genetic manipulation. |
